Lactobacillus casei, an oral bacterium prevalent in carious lesions, is one of only two lactic acid species capable of utilizing the pentitols xylitol (Xtl) and ribitol (Rtl) as growth substrates. Such polyols occur naturally in plants and are used as artificial sweeteners in sugar-free products. While cariogenic streptococci cannot metabolize these polyols, L. casei and Enterococcus avium are capable of producing acid from them. Studying the enzymes involved in metabolism of Rtl and Xtl is therefore pertinent to oral health. L. casei transports Rtl and Xtl via a specific phosphotransferase system (PTS). The Rtl and Xtl pathways each comprise a membrane-bound permease, a soluble III-ribitol or III-xylitol, and a pentitol-5-phosphate dehydrogenase. It is our goal to characterize the structure and function of the protein components of the Rtl and Xtl PTS pathways, and to study their underlying mechanisms of genetic regulation. Our approaches focus on Rtl-5-P dehydrogenase (RtlA), the rtl operon repressor protein (RtlR), and III-xylitol. RtlA was cloned from and L. casei DNA library by hybridization and immunological screening. RtlA shares sequence identity with several NADH-dependent dehydrogenases, but is not related to the functionally similar dehydrogenases of the hexitol PTS of enteric bacteria. Another gene, designated rtlR, was identified and potentially encodes a repressor protein. RtlR shares identity with members of the DeoR family of transcriptional repressors, including regulators of other PTS operons such as the Streptococcus mutans and Lactococcus lactis lactose PTS. The genes encoding the Rtl permease and III-ribitol were not present within the cloned L. casei DNA fragment carrying rtlA and rtlR. The transcriptional initiation sites of rtlA and rtlR were identified and permitted the localization of the promoters for these genes. A bacteriophage clone isolated from an L. casei library directs the synthesis of a polypeptide which cross-reacts with anti-III-xylitol antisera. The genetic analysis of this clone is in progress. We propose to investigate the regulation of the rtl operon and to explore the relatedness of the E. avium and L. casei Rtl and Xtl PTS.